When recombinant DNA techniques were developed, expectations were high regarding large-scale protein production using genetically modified bacteria. The majority of commercially attractive proteins, however, necessarily undergo post-translational modifications before they can become biologically active proteins. Hence, animal cells are now more frequently used to produce recombinant proteins.
Among animal cells, insect cells are of growing importance for the production of recombinant proteins. A convenient and versatile baculovirus vector system using insect cells has been developed. Information on the physiology of insect cells is rather scarce, however, vaccines produced via baculovirus recombinant techniques are generally well accepted. An example is the use of a baculovirus—expressed gp 160 envelope protein of human immunodeficiency virus type I as a possible AIDS vaccine in clinical trials.
Until now, large-scale production of baculovirus-expressed proteins in insect cells was limited to bioreactors of up to about 10 liters. Scale-up suspension cultures offer the best possibility. In large-scale production (see Tramper et al., Rec. Adv. Biotech., 1992, 263-284; Power and Nielsen, Cytotechnology 20: 209-219, 1996), special emphasis should be given to factors influencing cell growth and virus production. Variations in such factors greatly influence the final level of recombinant protein production.
Baculoviruses are characterized by rod-shaped virus particles which are generally occluded in occlusion bodies (also called polyhedra). The family Baculoviridae can be divided in two subfamilies: the Eubaculovirinae comprising two genera of occluded viruses—nuclear polyhedrosis virus (NPV) and granulosis virus (GV)—and the subfamily Nudobaculovirinae comprising the non-occluded viruses. The cell and molecular biology of Autographa californica (Ac)NPV has been studied more in detail.
Many proteins have been expressed in insect cells infected with a recombinant baculovirus encoding that protein. Encoding means that such viruses are provided with a nucleic acid sequence encoding a heterologous protein and often are further provided with regulating nucleic acid sequences, such as a promoter. Most often, the polyhedrin promoter is used to express a foreign gene but the p10 promoter is equally well suited and used as well.
Several cell-lines are available for infection with recombinant baculovirus. The cell-line SF-21 was derived from ovarian tissue of the fall armyworm (Spodoptera frugiperda). A clonal isolate, SF-9, available from the American Type Culture Collection (CRL 1711), is more or less a standard cell-line for in vitro production of recombinant virus and is said to be superior in producing recombinant virus. Other cell-lines are, for example, the Hi-Five cell-line and the Tn-368 and Tn-368A cell-lines obtained from the cabbage looper (Trichoplusia ni). The most widely used media in which insect cells grow include TNM-FH, BML-TC/10, and IPL-41. These media are usually supplemented with more or less defined components, such as mammalian sera, in particular, fetal calf serum. Serum replacements have also been applied to insect-cell culture, and serum-free media, such as Ex-cell 400™ and Sf900 are commercially available.
Insect cells, in general, grow on solid supports as well as in suspension, but are reported to give higher yields of virus when grown on solid supports. Infection is most efficient when cells are infected in the exponential growth phase. The amount of polyhedra and virus produced per cell, however, does not vary significantly between cells infected during different stages in the cell cycle. Cell density has a great influence on virus production. Insect cells can show a form of contact inhibition resulting in reduced virus production at higher cell densities.
The initial multiplicity of infection (“m.o.i.” or “MOI”), which is the number of infectious viruses per cell, generally influences both the fraction of infected cells and the number of polyhedra per cell at the end of infection. Optimal m.o.i. for virus production is generally considered to be at around 20-30. In a study (Licari and Bailey, Biotech. Bioeng., 37:238-246, 1991) of a recombinant baculovirus expressing β-galactosidase, Sf-9 cells were infected with m.o.i. values between 0 and 100. The β-galactosidase yield increased and cell density decreased with increasing m.o.i. It is generally thought that increasing or decreasing m.o.i. has only a limited affect on the maximum achievable yield of a recombinant protein per infected cell. Choosing low m.o.i., however, allows reduction of virus stock needed for infection and minimizes the risk of the generation of defective interfering particles of baculovirus. If a batch culture of insect cells is infected at high m.o.i. (>5), the ensuing infection process will be essentially synchronous, i.e., all cells will go through the infection cycle simultaneously. When cells are infected at an m.o.i. <5 in a batch culture, the culture will no longer be synchronous. The culture will initially be composed of non-infected cells and cells at different points in their individual infection cycle until all cells have been infected and the production of wanted protein comes to an end. In general, in such cultures the production levels are much lower. The culture behavior is the combined behavior of the individual cells that are each in a different phase of production, thus, the suboptimal production levels. In a continuous culture, non-infected cells are added continuously and the culture will obviously be asynchronously infected.
Through designing mathematical models, it is thought possible to predict complex behaviors such as those observed when infecting cells at low m.o.i. or when propagating virus in a continuous culture system. A purely empirical analysis of the same phenomena is considered very difficult, if not impossible. At present, three models are known: the Licari & Bailey, the de Gooijer and the Power & Nielsen model. These are, despite their complexity and the effort that has gone into developing them, all first generation models, postulating about the behavior of baculoviruses expressing a model recombinant protein (β-galactosidase) expressed under control of the polyhedrin promoter. They summarize, to a large extent, our present quantitative understanding of the interaction between baculovirus and insect cells, when looked upon as a black box system, with disregard to DNA and RNA accumulation and the infection cycle. The binding and initial infection processes are still quantitatively poorly understood and further work in this area is much needed.
The baculovirus expression system offers a powerful tool for recombinant protein production. Several cell-culture configurations can be used for large-scale production. These systems, however, need further optimization to take full advantage of their potential. For commercial application, large-scale and low-cost production is pivotal. Polyhedra-production systems reported in large-scale cell cultures should be dramatically improved to meet the commercial demands for a price-competitive product.